title: Molecular basis of dimethylglycine dehydrogenase deficiency associated with pathogenic variant H109R.
abstract: Dimethylglycine dehydrogenase (DMGDH) is a mitochondrial matrix flavoprotein that catalyses the demethylation of dimethylglycine to form sarcosine, accompanied by the reduction of the covalently bound FAD cofactor. Electron-transfer flavoprotein reoxidizes the reduced flavin and transfers reducing equivalents to the main mitochondrial respiratory chain through the enzyme ETF-ubiquinone oxidoreductase. DMGDH plays a prominent role in choline and 1-carbon metabolism. We have expressed the mature form of human DMGDH and the H109R variant identified in a DMGDH-deficient patient as N-terminally His(6)-tagged proteins in E. coli. The enzymes were purified to homogeneity by nickel affinity and anion exchange chromatography. The presence of FAD in the wild-type enzyme was confirmed by spectrophotometric analysis. The H109R variant, however, had only 47% of the wild-type level of bound flavin as expressed in E. coli, indicating its reduced affinity for FAD As previously described for rat enzyme studies, the wild-type human enzyme exhibited two K (m) values for N,N-dimethylglycine (K (m1) = 0.039 +/- 0.010 mmol/L and K(m2) = 15.4 +/- 1.2 mmol/L). The addition of 4 micromol/L tetrahydrofolate resulted in a slight decrease in specific activity and a substantial decrease in K (m2) (1.10 +/- 0.55 mmol/L). The flavinated H109R variant protein exhibited a 27-fold decrease in specific activity and a 65-fold increase in K (m), explaining its pathogenicity. Additionally, the current expression system represents a significant improvement over a previously described rat DMGDH expression system and will enhance our ability to further study this important metabolic enzyme.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
authors.forename: R P
authors.initials: RP
authors.lastname: McAndrew
authors.forename: J
authors.initials: J
authors.lastname: Vockley
authors.forename: J-J P
authors.initials: JJ
authors.lastname: Kim
mesh_terms: D002852:Chromatography, Ion Exchange / Q000379:methods; D050893:Dimethylglycine Dehydrogenase / Q000737:chemistry / Q000172:deficiency* / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005420:Flavoproteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008661:Metabolism, Inborn Errors / Q000175:diagnosis / Q000235:genetics*; D008956:Models, Chemical; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D050892:Sarcosine Dehydrogenase / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods
chemical_list: D005420:Flavoproteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D050892:Sarcosine Dehydrogenase; D050893:Dimethylglycine Dehydrogenase; D010100:Oxygen